How to predict implantation? No correlation between antigen G-concentrations

How to predict implantation? No correlation between
embryonic aneuploidy and soluble human leukocyte
antigen G-concentrations
Carolyn B. Coulam, M.D.,a,b Roumen G. Roussev, M.D., Ph.D.,b Sveltana Lerner, M.S.,c
Zev Zlatopolsky, B.S.,c Yuri Ilkevitch, Ph.D.,c and Ilan Tur-Kaspa, M.D.c,d
a
Rinehart Center for Reproductive Medicine, Evanston; and b CARI Reproductive Institute, c Reproductive Genetics Institute,
and d Institute for Human Reproduction (IHR), Chicago, Illinois
Objective: To determine if soluble human leukocyte antigen-G (sHLA-G) concentrations in spent culture media
may assist in identifying the normal embryo for implantation.
Design: Prospective blinded comparative study.
Setting: Reproductive genetic and reproductive medicine centers.
Patient(s): One hundred and sixteen embryos obtained from eight patients undergoing in vitro fertilization (IVF)
with preimplantation genetic diagnosis (PGD).
Intervention(s): Culture media obtained 2 days after fertilization were analyzed for sHLA-G concentrations using
an enzyme-linked immunosorbent assay (ELISA) assay. A sHLA-G concentration of R1.9 mIU/mL was considered a positive predictor for successful implantation. Polar bodies and blastomeres from day-3 embryos were tested
by PGD for 5 to 11 chromosomes: 8, 9, 13, 15, 16, 17, 18, 21, 22, X, and Y.
Main Outcome Measure(s): The results of the sHLA-G concentrations were compared with the results of the PGD
analyses.
Result(s): We found an sHLA-G concentration R1.9 mIU/mL in 48% (56 out of 116) and normal PGD results in
52% (57 out of 116) of embryos. Of the embryos with normal PGD results, 46% (26 out of 57) had sHLA-G concentrations R1.9 mIU/mL. Among the embryos with sHLA-G R1.9 mIU/mL, 46% (26 out of 56) had normal PGD
results, and 21% of embryos displayed both normal PGD results and sHLA-G R1.9 mIU/mL.
Conclusion(s): No correlation between concentrations of sHLA-G in embryo culture media and PGD results of an
embryo’s aneuploidy were observed. (Fertil Steril 2009;91:2408–13. 2009 by American Society for Reproductive Medicine.)
Key Words: Soluble HLA-G, preimplantation genetic screening, PGD, implantation, embryo selection, aneuploidy
Can we predict which embryo will implant and result in
a pregnancy of a fetus with normal karyotype? Answering
this question is becoming even more important with the current trend for single embryo transfer because of the high incidence of multiple gestations and resultant increase in
preterm births and perinatal morbidity after in vitro fertilization (IVF) and embryo transfer compared with natural conceptions (1). Although single embryo transfers result in
a marked decrease in multiple births, it is associated also
with a decrease in pregnancy rates per cycle (2). The
approach to improving pregnancy rates after single embryo
transfer has focused on selection of embryos most likely to
implant and end in successful live birth. Methods currently
being employed to select embryos include embryo morphology (3), preimplantation genetic diagnosis (PGD) (4, 5), and
Received January 22, 2008; revised and accepted March 31, 2008; published online April 25, 2009.
C.B.C. has nothing to disclose. R.G.R. has nothing to disclose. S.L. has
nothing to disclose. Z.Z. has nothing to disclose. Y.I. has nothing to disclose. I.T-K. has nothing to disclose.
Supported in part by an independent research grant from Ferring Pharmaceutical Company.
Reprint requests: Carolyn Coulam, M.D., 2500 Ridge Ave, Suite 200,
Evanston, IL 60201 (FAX: 847-869-7782; E-mail: cbcoulam@aol.com).
2408
measurement of soluble human leukocyte antigen-G (sHLAG) concentrations in embryo culture media (6–9). Embryo
morphology is currently the most commonly used method
of selecting embryos worldwide, but its predictive value by
itself is limited (10, 11). Recent publications have suggested
that PGD (4, 5) and embryo culture media determination of
sHLA-G concentration (6–9) have been helpful in selecting
embryos that result in an enhanced live birth rate than nonselection. The question then arises as to relationship between
the results of PGD and concentration of sHLA-G in embryo
culture media. Our study examined whether sHLA-G can assist in identifying the normal embryo for implantation.
MATERIALS AND METHODS
One hundred and sixteen embryos out of 133 from eight patients undergoing IVF with PGD for treatment for infertility
or to conceive with a healthy baby comprised the study population. All embryos were biopsied for PGD. In four patients,
PGD was performed only for aneuploidy; in other
four patients, PGD was for aneuploidy and for different single-gene disorders: fragile-X syndrome, spinal muscular atrophy, hereditary nonpolyposis colorectal cancer type I, and
Fanconi anemia as well as HLA typing. The polar bodies
Fertility and Sterility Vol. 91, No. 6, June 2009
Copyright ª2009 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/09/$36.00
doi:10.1016/j.fertnstert.2008.03.081
and blastomere biopsies for PGD were approved by the institutional review board of Reproductive Genetics Institute, and
all patients signed informed consent documentation.
Preimplantation Genetic Diagnosis (PGD) Procedure
The PGD for aneuploidy was performed by fluorescence in
situ hybridization (FISH) analysis of polar bodies and blastomeres, using commercially available chromosome-specific
probes (Abbot, Downers Grove, IL) (5) or by polymerase
chain reaction (PCR). Polar bodies and blastomeres from
day-3 embryos were tested by PGD for 5 to 11 chromosomes:
8, 9, 13, 15, 16, 17, 18, 21, 22, X, and Y. The PCR was used
for genetic analysis of polar bodies and blazoners for singlegene disorders or HLA typing. We performed PCR aneuploidy testing by adding primers for chromosome-specific
microsatellite markers to the multiplex PCR protocols
worked out for a specific genetic disorder or HLA typing.
The primers for PCR-based aneuploidy testing used for identification of the copy number of chromosomes 13, 16, 18, 21,
22, X, and Y were recently described by Rechitsky et al. (12).
Determination of sHLA-G
The level of sHLA-G in embryo culture media was detected
by using a specific enzyme-linked immunosorbent assay
(ELISA) kit (EXBIO/BioVendor, Praha, Czech Republic).
The capture antibody is a specific anti-sHLA-G monoclonal
clone MEM-G9, and the detecting antibody is anti-b2-microglobulin. The embryo culture media was added in each single-well as 50-mL droplets without oil contamination. The
standard sHLA-G was diluted to give a calibration curve
within the range of expected sHLA-G secretion in embryo
culture media. The standard curve and extrapolation of
each sample’s readings were made with TableCurve2D
(Systat Software, Inc, Richmond, CA) software.
Statistical Analysis
The physicians performing embryo transfer, the embryologists, and the geneticists analyzing the PGD results were
blinded to the sHLA-G testing. The determination of
sHLA-G was performed blindly to the results of the PGD.
The frequencies of normal and abnormal PGD results and
sHLA-G results were compared using a 2 2 contingency
table with Fisher’s exact test (InStat; GraphPad Software,
San Diego, CA). The correlation between PGD results and
sHLA-G culture media concentrations was tested using linear
regression. A two-tailed P<.05 was considered statistically
significant.
RESULTS
A total of 133 embryos were generated from eight women
(mean age: 32.4 years SD) undergoing IVF with PGD.
Of these, 17 embryos were not tested for aneuploidy because
they were affected by the mutation or did not develop to blastocyst stage and thus were determined unsuitable for transfer,
Fertility and Sterility
which left 116 embryos (87%) with aneuploidy results that
were included in the study. Of the 116 embryos, 57 (49%)
had normal PGD results, and 59 (51%) had abnormal results.
A list of the abnormal results is shown in Table 1. The
sHLA-G culture media concentrations were R1.9 mIU/mL
in 56 (47%) and <1.9 mIU/mL in 61 (53%).
Of the 57 embryos displaying normal PGD results, 26
(46%) had sHLA-G culture media concentrations R1.9
mIU/mL, and 31 (54%) had concentrations <1.9 mIU/mL
(Fig. 1A). When considering the 56 embryos that secreted
R1.9 mIU/mL sHLA-G, 26 (46%) had normal PGD results,
and 30 (54%) had abnormal results (see Fig. 1B).
Linear regression revealed no correlation between PGD
results and sHLA-G concentrations in the embryo culture
media, with correlation coefficient r ¼ 0.077 and r2 ¼
0.006, P¼.4 (Fig. 2).
The combination of normal PGD results and sHLA-G concentrations of R1.9 mIU/mL in their culture media was observed in 24 of the 116 embryos studied (21%). Two babies
were born with sHLA-G of 1.9 mIU/mL, and two biochemical pregnancies also occurred with sHLA-G of 1.5 and
1.6 mIU/mL.
DISCUSSION
Our data show no correlation between PGD results of an embryo’s aneuploidy and concentrations of sHLA-G in the embryo culture media. This finding suggests that chromosomal
TABLE 1
Chromosomal abnormalities found in 116
embryos undergoing preimplantation genetic
screening.
Chromosomal abnormality
N
þ9
13
þ13–22
16
þ16
18
þ18
21
þ21
22
þ22
21–22
þ21þ22
Chaotic
XO
Polyploid
Haploid
1
1
1
2
4
1
5
1
11
4
4
2
2
9
1
4
1
Coulam. No correlation of aneuploidy and sHLA-G. Fertil Steril 2009.
2409
FIGURE 1
FIGURE 2
Percentage of PGD results and sHLA-G culture
media concentrations. (A) Of the 56 embryos that
secreted R1.9 mIU/mL sHLA-G, 23 (41%) had
normal PGD results, and 33 (59%) had abnormal
results. (B) Of the 57 embryos displaying normal
PGD results, 26 (46%) had sHLA-G culture media
concentrations R1.9 mIU/mL, and 31 (54%) had
concentrations <1.9 mIU/mL.
Linear regression analysis showing no correlation
between PGD results and sHLA-G concentrations in
embryo culture media with correlation coefficient
r ¼ 0.077 and r2 ¼ 0.006, P¼ .4.
r=0.077
R2=0.006
P=0.4
F=0.684
KARYOTYPE
A
Abnormal 2
%
100
80
60
sHLA-G>1.9miu/ml
sHLA-G<1.9miu/ml
40
Normal 1
20
0
0
1
2
3
4
5
6
7
8
sHLA-G miu/ml
Normal
Coulam. No correlation of aneuploidy and sHLA-G. Fertil Steril 2009.
Abnormal
Karyotype
B%
100
80
60
Karyotype
Normal
Abnormal
40
20
0
sHLA-G≥1.9
sHLA-G<1.9
miu/ml
Coulam. No correlation of aneuploidy and sHLA-G. Fertil Steril 2009.
abnormality and decreased secretion of sHLA-G by embryos
are independent markers of risk for lack of implantation. Soluble HLA-G concentrations in spent culture media cannot
identify a normal embryo for implantation without aneuploidy testing by PGD. Because abnormal embryos do implant, it is not surprising that no correlation was found. One
possibility that needs further investigation is to identify level
of sHLA-G that may predict which of the normal embryos
available for transfer would implant.
Although both PGD results (4, 5) and diminished concentrations of sHLA-G in embryo culture media (6–9) have been
shown to be risk factors for unsuccessful implantation, each
by itself has limitations. By selecting only chromosomally
normal embryos for transfer, PGD was expected to improve
IVF outcome. To date, over 20,000 PGD procedures have
been performed worldwide (13) and controversy still exists
regarding its efficacy (13–15). Indeed, one recently published
large randomized study indicated that PGD was detrimental
to pregnancy outcome (16). Three explanations have been
2410
Coulam et al.
put forward to address this controversy. The first explanation
involves the number of chromosomes studied when performing PGD. The number of aneuploidic embryos demonstrated
increases with the number of chromosomes investigated.
Technical limitations of using FISH restrict the number of
chromosomes that can be screened. Most laboratories offering PGD services currently examine 5 to 12 chromosomes
per embryo. Screening for aneuploidies within the chromosomes most often found to be abnormal in prenatal samples
has been successful in reducing the number of such aneuploidies (17), but a significant improvement in embryo
implantation could not be shown (18). This observation suggests that aneuploidy for additional untested chromosomes is
occurring, compromising the embryo implantation rate. It is
hoped that newer techniques using microarray comparative
genomic hybridization (CGH) (19) or gene chips (20) will
help to address the issue of the number of chromosomes
investigated for PGD.
A second explanation for the discrepant results describing
pregnancy and live birth rates after PGD has been a substandard application of PGD techniques (21). Munne et al. (21)
stated that, without expert biopsy sampling leading to minimal embryo damage and expert fixation methods leading to
low error rates, the benefits of PGD cannot be realized. A
third explanation for the lack of improvement of pregnancy
and live birth rates after PGD involves the major pitfall of
aneuploidy screening with a single blastomere biopsy and
the existence of mosaicism (22–24). Chromosomally normal
and abnormal blastomeres can coexist within the same earlycleavage-stage embryo (22). A recent report described the
rate of mosaicism among day-3 embryos to be 50% (23),
and a discordance rate of over 70% was noted when two blastomeres were biopsied from the same embryo on day 3 and
No correlation of aneuploidy and sHLA-G
Vol. 91, No. 6, June 2009
analyzed for eight chromosomes (25). Because of these findings, the Practice Committee of the American Society for Reproductive Medicine has recently reported an opinion that
‘‘available evidence does not support the use of Preimplantation Genetic Screening as currently performed to improve
live birth rates in patients with advanced maternal age, previous implantation failure or a history of recurrent pregnancy
loss’’ (26). Because in our study the polar bodies were also
tested, the possible mosaicism is very low.
HLA-G is a nonclassic major histocompatability (MHC)
class I antigen (27) that has been proposed to have a pivotal
role in the protection of the allogenic embryo from the maternal immune system (28). In 1996, Jurisicova first reported
HLA-G heavy chain mRNA was present in 40% of blastocysts, in some cleavage stage embryos, and in unfertilized
oocytes (29). In concordance with mRNA data, a similar proportion of embryos stain positive for HLA-G immunohistochemistry (30). Alternative splicing of its primary transcript
gives rise to both membrane-bound and soluble isoforms of
HLA-G (31). Although interaction between soluble forms
of HLA G (sHLA G) secreted by the trophoblasts and uterine
lymphocytes in the decidua has been proposed to be a major
player in the induction of immunotolerance for the invading
blastocyst (28), the natural killer (NK) cell receptors responsible for HLA-G recognition have remained controversial.
Initial studies suggested that killer-cell immunoglobulinlike receptor (KIR) molecules on NK cells interact with
HLA-G (32). However, these findings have not been confirmed by other independent laboratories (33, 34). Until recently, it seemed that NK cell recognition of HLA-G by
peripheral blood or decidual NK cells could be explained
by the CD94/NKG2A receptor interacting with HLA-E containing and HLA-G–derived leader peptide in the antigenbinding groove or, in a minority of cases, by direct HLA-G
recognition by immunoglobulin-like transcript 2 (ILT2) on
an NK cell subset (33, 34). Ponte et al. (35) and Rajagopalan
and Long (36) have more recently implicated a receptor encoded by the KIR family in HLA-G recognition, designated
KIR2DL4. Rajagopalan and Long (36) have shown by genetic transfer that the KIR2DL4 molecule inhibits NK cell lysis of target cells expressing HLA-G. Both Rajagopalan and
Long (36) and Cantoni et al. (37) have demonstrated direct
binding of KIR2DL4 fusion proteins to HLA-G, confirming
the specificity of this interaction. Furthermore, Ponte et al.
(35) reported that KIR2DL4 is expressed on a substantial proportion of NK cells in the decidua during the first trimester of
pregnancy. Soluble HLA-G suppresses NK cytotoxicity (38),
stimulates interferon-g and tumor necrosis factor a production by NK cells (39), and induces apoptosis of CD8þ T cells
(40) as well as stimulates production of transforming growth
factor b-1 (TGF-b1) by activated macrophages (41). Taken
together, these observations suggest that the function of
HLA-G is to modulate cytokine secretion to induce immunotolerance; however, nonimmune functions to allow for successful embryo implantation and pregnancy maintenance,
including controlling trophoblast invasion and contributing
to vascular remodeling of spiral arteries, have also been proFertility and Sterility
posed (42). Because the gene for HLA-G is located on chromosome 6 and aneuploidy for chromosome 6 only occurs in
6% (12), this does not explain our results.
Although determination of sHLA-G concentrations in embryo culture media (6–9) has been shown to be helpful in selecting embryos that result in an enhanced implantation rate
compared with nonselection (6–9), it, too, has its limitations.
Similar to the findings in our study, a previous report found
that half of the embryo culture media tested had sHLA-G
concentrations R1.9 IU/mL and half had <1.9 IU/mL (9).
However, only half of the embryos with HLA-G concentrations R1.9 mIU/mL implanted after they were transferred
into the uterus, giving a positive predictive value for implantation of 50% (9). That not all embryos whose culture media
had sHLA-G concentrations R1.9 mIU/mL resulted in pregnancy suggests that sHLA-G is essential for but not sufficient
for implantation to occur.
In other words, not all embryos fail to implant for the same
reason. The goal would be to identify multiple risk factors for
implantation failure and to test for multiple analytes. By identifying the embryo most likely to implant and lead to a live
birth, the ultimate goal of single embryo transfer and singleton birth for each cycle would be enhanced. Achieving this
goal would answer a number of questions regarding the medical and economic concerns currently plaguing assisted
reproductive procedures.
Human reproduction is an inefficient process, with only
22.8% of conceptive matings resulting in live birth (43). Failure of implantation accounts for 75% of conceptions that are
lost (44) and is the most common cause of lack of successful
pregnancy after IVF and embryo transfer. Among infertile
women, implantation rates of approximately 15% per embryo
transferred after IVF render the process of assisted reproduction inefficient (45). To try to adjust for the inefficiency, multiple embryos are transferred, increasing the risk of multiple
gestations. Multiple gestations are associated with increased
maternal mortality (46) and perinatal morbidity and mortality
(47). The risks of preterm birth and perinatal mortality are increased after IVF as a result of increased multiple gestations
(1, 48). The only way to decrease these risks is to transfer single embryos. A number of studies have shown the success of
decreasing the multiple gestation rate with (2, 49) and without (50–52) decreasing the pregnancy rate per cycle in both
younger (<36 years) (2, 49–52) and older (36 to 39 years)
(53) women, especially if frozen embryo transfers are considered in the calculation (49, 51, 54). Single blastocyst stage
transfers resulted in a statistically significantly higher delivery rate than single cleavage stage embryo transfers (32%
vs. 22%) (55). Although all studies demonstrated a decrease
in multiple gestations with single embryo transfers, concern
has been expressed that the twinning rate is reduced at the expense of declining pregnancy rates, a need for more treatment
cycles, a potential delay in treatment success, and higher
treatment costs (56). However, economic studies have shown
that transfer of a single top quality embryo can be less expensive than double embryo transfers (46, 57, 58). It is, therefore,
2411
important to be able to select the top quality or single embryo
most likely to succeed.
Of interest was the observation that only 21% of embryos
tested displayed both normal PGD results and sHLA-G concentrations R1.9 mIU/mL in their respective culture media.
Epidemiologic data suggest that 20% of women conceive
each cycle, even when couples are fertile (59). Because
abnormal embryos do implant, prospective clinical trials
are necessary to answer the question of the effectiveness of
embryo selection using both PGD for aneuploidy and
sHLA-G.
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